Materials and MethodsAs in the study of the previously determined MscL structure (S1), multiple homologs of MscS [encoded by the yggb gene (S2)] were identified by BLAST searching of the NCBI genome database. Ten homologs from prokaryotes and Archaea were identified and subsequently cloned. The channels were subcloned into expression vectors (pET system, Novagen) and expression screening was carried out. Cells expressing sufficient channels to be identified by Western blotting were subjected to extensive detergent screening utilizing ~50 detergents (Anatrace, Sigma, Aldrich) where both the ability of the channels to be extracted out of the membrane and the ability to remain as a homo-oligomer were determined. Subsequent large-scale expressions, extraction and purification produced sufficient amounts of protein for three channels (E. coli, B. subtilis and C. tepidum) for crystallization trials. Each of these channels was produced recombinantly (vector pet28b, Novagen) in 50-liter fermenter growths in a modified Terrific Broth media containing 1% glucose and 0.4% glycerol. Protein expression was initiated by the addition of 2% lactose and 2 mM IPTG for 2-4 hours, resulting in ~1.5 kg of wet cells. To obtain phase information, selenomethionine-derivatized protein was purified from cells grown in a modified M9 media containing 50 mg/l selenomethionine, and the remaining amino acids at 40 µg/l. Extraction of the E. coli MscS was carried out using sonication and solubilization with 1% Foscholine-14. Ni-affinity chromatography, anion exchange, and size exclusion chromatography in the presence of 0.05% Foscholine-14 were used to purify the protein to homogeneity. The apparent molecular mass of the protein, as indicated by size-exclusion chromatography, was in excess of 200 kD, similar to that reported for recombinant MscS by Sukharev (S3). Crystals were obtained with 10-15 mg/ml MscS by hanging drop vapor diffusion with 100 mM pH 7.2 Hepes buffer, 150 mM Na-formate, 8% glycerol, and 16% PEG-3350 as the precipitant. Crystals grew to ~200 µm in each dimension, and were assigned to space group P4 3 2 1 2 (a = b = 184.7 Å, c = 260.7 Å) with one MscS oligomer in the asymmetric unit (corresponding to ~71% solvent content). Only residues in the extramembrane (water-soluble) regions of MscS participated in lattice contacts.

The chemosensory pathway of bacterial chemotaxis has become a paradigm for the two-component superfamily of receptor-regulated phosphorylation pathways. This simple pathway illustrates many of the fundamental principles and unanswered questions in the field of signaling biology. A molecular description of pathway function has progressed rapidly because it is accessible to diverse structural, biochemical, and genetic approaches. As a result, structures are emerging for most of the pathway elements, biochemical studies are elucidating the mechanisms of key signaling events, and genetic methods are revealing the intermolecular interactions that transmit information between components. Recent advances include (a) the first molecular picture of a conformational transmembrane signal in a cell surface receptor, (b) four new structures of kinase domains and adaptation enzymes, and (c) significant new insights into the mechanisms of receptor-mediated kinase regulation, receptor adaptation, and the phospho-activation of signaling proteins. Overall, the chemosensory pathway and the propulsion system it regulates provide an ideal system in which to probe molecular principles underlying complex cellular signaling and behavior.

No abstract

The transmembrane, homodimeric aspartate receptor of Escherichia coli and Salmonella typhimurium controls the chemotactic response to aspartate, an attractant, by regulating the activity of a cytoplasmic histidine kinase. The cytoplasmic domain of the receptor plays a central role in both kinase regulation and sensory adaptation, although its structure and regulatory mechanisms are unknown. The present study utilizes cysteine and disulfide scanning to probe residues Leu-250 through Gln-309, a region that contains the first of two adaptive methylation segments within the cytoplasmic domain. Following the introduction of consecutive cysteine residues by scanning mutagenesis, the measurement of sulfhydryl chemical reactivities reveals an ␣-helical pattern of exposed and buried positions spanning residues 270 -309. This detected helix, termed the "first methylation helix," is strongly amphiphilic; its exposed face is highly anionic and possesses three methylation sites, while its buried face is hydrophobic. In vivo and in vitro assays of receptor function indicate that inhibitory cysteine substitutions are most prevalent on the buried face of the first methylation helix, demonstrating that this face is involved in a critical packing interaction. The buried face is further analyzed by disulfide scanning, which reveals three "lock-on" disulfides that covalently trap the receptor in its kinaseactivating state. Each of the lock-on disulfides crosslinks the buried faces of the two symmetric first methylation helices of the dimer, placing these helices in direct contact at the subunit interface. Comparative sequence analysis of 56 related receptors suggests that the identified helix is a conserved feature of this large receptor family, wherein it is likely to play a general role in adaptation and kinase regulation. Interestingly, the rapid rates and promiscuous nature of disulfide formation reactions within the scanned region reveal that the cytoplasmic domain of the full-length, membranebound receptor has a highly dynamic structure. Overall, the results demonstrate that cysteine and disulfide scanning can identify secondary structure elements and functionally important packing interfaces, even in proteins that are inaccessible to other structural methods.The aspartate receptor of Escherichia coli and Salmonella typhimurium is representative of a large family of cell-surface receptors that regulate two-component signaling pathways, which are widespread in prokaryotic and eukaryotic organisms (1-9). These receptors contain two putative transmembrane helices per subunit and, in all cases tested, form stable homodimers that signal via a transmembrane conformational change. Chimeric receptors containing the sensory domain of the aspartate receptor and the regulatory domain of another family member are functional, suggesting that members of this receptor family use a conserved mechanism of transmembrane signaling to regulate cytoplasmic histidine kinase activity (10 -12). More generally, conformational transmembrane signals ma...

The transmembrane aspartate receptor of Escherichia coli and Salmonella typhimurium propagates extracellular signals to the cytoplasm, where its cytoplasmic domain regulates the histidine kinase, CheA. Different signaling states of the cytoplasmic domain modulate the kinase autophosphorylation rate over at least a 100-fold range. Biochemical and genetic studies have implicated a specific region of the cytoplasmic domain, termed the signaling subdomain, as the region that transmits regulation from the receptor to the kinase. Here cysteine and disulfide scanning are applied to the N-terminal half of the signaling subdomain to probe its secondary structure, solvent exposure, and protein-protein interactions. The chemical reactivities of the scanned cysteines exhibit the characteristic periodicity of an ␣-helix with distinct solvent-exposed and buried faces. This helix, termed ␣7, ranges approximately from residue 355 through 386. Activity measurements probing the effects of cysteine substitutions in vivo and in vitro reveal that both faces of helix ␣7 are critical for kinase activation, while the buried face is especially critical for kinase down-regulation. Disulfide scanning of the region suggests that helix ␣7 is not in direct contact with its symmetric partner (␣7) from the other subunit; presently, the structural element that packs against the buried face of the helix remains unidentified. Finally, a novel approach termed "protein interactions by cysteine modification" indicates that the exposed C-terminal face of helix ␣7 provides an essential docking site for the kinase CheA or for the coupling protein CheW.A fundamental question in signaling biology concerns the mechanism by which cell surface receptors regulate cytoplasmic kinases. Certain receptors activate their associated kinases by dimerization, but in other cases kinase activation is triggered by a transmembrane conformational change. One class of receptors that generates such conformational regulation is the large group of cell surface receptors that modulate histidine kinases in prokaryotic and eukaryotic two-component signaling pathways (reviewed in Refs. 1-9). The aspartate receptor of bacterial chemotaxis is representative of this diverse class. The aspartate receptor is one of the ligand specific dimeric receptors utilized by the related chemotaxis pathways of Escherichia coli and Salmonella typhimurium to recognize periplasmic attractant and repellent molecules. This receptor binds aspartate, an attractant, in the periplasm and propagates a signal across the bilayer to an associated histidine kinase in the cytoplasm. Receptor-kinase coupling is provided by the formation of a kinetically stable ternary complex between the cytoplasmic domain of the receptor, the coupling protein CheW, and the histidine kinase CheA (10, 11). The cytoplasmic phosphorylation cascade is initiated by receptorstimulated CheA autophosphorylation; subsequently, phosphoCheA becomes the substrate for a phospho-transfer reaction that phosphorylates one of two response regul...

Site-directed cysteine and disulfide chemistry is broadly useful in the analysis of protein structure and dynamics, and applications of this chemistry to the bacterial chemotaxis pathway have illustrated the kinds of information that can be generated. Notably, in many cases, cysteine and disulfide chemistry can be carried out in the native environment of the protein whether it be aqueous solution, a lipid bilayer, or a multiprotein complex. Moreover, the approach can tackle three types of problems crucial to a molecular understanding of a given protein: (1) it can map out 2° structure, 3° structure, and 4° structure; (2) it can analyze conformational changes and the structural basis of regulation by covalently trapping specific conformational or signaling states; and (3) it can uncover the spatial and temporal aspects of thermal fluctuations by detecting backbone and domain dynamics. The approach can provide structural information for many proteins inaccessible to high-resolution methods. Even when a high-resolution structure is available, the approach provides complementary information about regulatory mechanisms and thermal dynamics in the native environment. Finally, the approach can be applied to an entire protein, or to a specific domain or subdomain within the full-length protein, thereby facilitating a divide-and-conquer strategy in large systems or multiprotein complexes.Rigorous application of the approach to a given protein, domain, or subdomain requires careful experimental design that adequately resolves the structural and dynamical information provided by the method. A full structural and dynamical analysis begins by scanning engineered cysteines throughout the region of interest. To determine 2° structure, the solvent exposure of each cysteine is determined by measuring its chemical reactivity, and the periodicity of exposure is analyzed. To probe 3° structure, 4° structure, and conformational regulation, pairs of cysteines are identified that rapidly form disulfide bonds and that retain function when induced to form a disulfide bond in the folded protein or complex. Finally, to map out thermal fluctuations in a protein of known structure, disulfide formation rates are measured between distal pairs of nonperturbing surface cysteines. This chapter details these methods and illustrates applications to two proteins from the bacterial chemotaxis pathway: the periplasmic galactose binding protein and the transmembrane aspartate receptor.

Therapeutic proteins contain a large number of post-translational modifications, some of which could potentially impact their safety or efficacy. In one of these changes, pyroglutamate can form on the N terminus of the polypeptide chain. Both glutamine and glutamate at the N termini of recombinant monoclonal antibodies can cyclize spontaneously to pyroglutamate (pE) in vitro. Glutamate conversion to pyroglutamate occurs more slowly than from glutamine but has been observed under near physiological conditions. Here we investigated to what extent human IgG2 N-terminal glutamate converts to pE in vivo. Pyroglutamate levels increased over time after injection into humans, with the rate of formation differing between polypeptide chains. These changes were replicated for the same antibodies in vitro under physiological pH and temperature conditions, indicating that the changes observed in vivo were due to chemical conversion not differential clearance. Differences in the conversion rates between the light chain and heavy chain on an antibody were eliminated by denaturing the protein, revealing that structural elements affect pE formation rates. By enzymatically releasing pE from endogenous antibodies isolated from human serum, we could estimate the naturally occurring levels of this post-translational modification. Together, these techniques and results can be used to predict the exposure of pE for therapeutic antibodies and to guide criticality assessments for this attribute.

The 25 rapidly formed and 14 functional disulfide bonds identify helix-helix contacts and their register in the full-length, membrane-bound receptor-kinase complex. The results reveal an extended, rather than compact, domain architecture in which the observed helix-helix interactions are best described by a four-helix bundle arrangement. A cluster of six lock-on disulfide bonds pinpoints a region of the four-helix bundle critical for kinase activation, whereas the signal-retaining disulfides indicate that signal-induced rearrangements of this region are small enough to be accommodated by disulfide-bond flexibility (< or = 1.2 A). In the absence of bound kinase, helix packing within the cytoplasmic domain is highly dynamic.